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1.
J Biomed Mater Res B Appl Biomater ; 112(1): e35330, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37737549

RESUMO

Enhanced mucosal sealing around titanium implants can reduce complications such as peri-implantitis. The present study aims to investigate the mucosal healing at the early stage around the protease activated receptor 4-agonist peptide (PAR4-AP)- or perpendicularly protruded type I collagen (pCol)-treated titanium implants. A total of 72 implants were placed in 36 rats in the study. Following extractions, two tissue-level implants among the following three different surfaces, PAR4-AP-coated (PAR4 group, n = 24), pCol-treated (pCol group, n = 24) and non-treated (control group, n = 24) ones, were placed in the maxillae of each rat based on a split-mouth design. The specimens retrieved at 8 h (n = 8 per group), 3 days (n = 8 per group), and 2 weeks (n = 8 per group), were immunostained and tissue-cleared, and the signals of laminin-5 and collagen fibers were observed under multiphoton microscopy. Statistical analyses were performed using linear mixed model with post hoc tests to compare differences between the groups. While there was no intergroup difference at 8 h, the laminin-5 at 3 days was more abundant near the PAR4-group-surface, and its area was significantly larger in the PAR4 group (0.0204 ± 0.0194 mm2 ) than the control (0.0019 ± 0.0025 mm2 , p = .001) and pCol (0.0023 ± 0.0022 mm2 , p < .001) groups. The pCol group showed a significantly larger area of collagen fibers (0.0230 ± 0.0148 mm2 ) compared to the control (0.0035 ± 0.0051 mm2 , p = .002) and PAR4 (0.0031 ± 0.0057 mm2 , p < .001) groups at 3 days. At 3 days and 2 weeks, the collagen fiber orientation of the pCol group showed a more perpendicular manner compared to the control and PAR4 groups. The signal of basal lamina and collagen fibers were stronger around the PAR4-AP- and pCol-treated titanium surfaces, respectively during the early healing stage. This could have implications for improved mucosal sealing around dental implants, potentially reducing complications such as peri-implantitis.


Assuntos
Implantes Dentários , Peri-Implantite , Ratos , Animais , Colágeno Tipo I/farmacologia , Titânio/farmacologia , Propriedades de Superfície , Peptídeos , Receptores Ativados por Proteinase
2.
Materials (Basel) ; 15(13)2022 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-35806723

RESUMO

Soft tissue sealing around zirconia (ZrO2) abutment is critical for the long-term stability of dental implants. The goal of the study is to develop a strong basal lamina (BL)-mediated epithelial attachment to ZrO2 via a novel physicochemical immobilization method. An electrophoretic fusion (EPF) method was applied to fuse a phosphonic acid (PA) linker to ZrO2 discs. Bindings of the PA linker and the following protease activated receptor 4 (PAR4) were verified by Fourier-transform infrared spectroscopy (FITR). Then, ZrO2 discs were doped in platelet-rich plasma (PRP). Platelet-derived growth factor (PDGF) was measured to assess platelet activation. PRP-doped discs were subsequently co-cultured with human gingival epithelial cells (OBA9) to evaluate establishment of basal lamina-mediated epithelial attachment. The EPF method achieved robust immobilization of the PA linker and PAR4 onto the ZrO2 surface. The resultant PAR4-coupled ZrO2 successfully induced platelet aggregation and activation. Furthermore, a BL-mediated epithelial attachment was established. The results are significant for clinical application to minimize the risk of developing peri-implant diseases.

3.
Altern Lab Anim ; 49(3): 83-92, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34218686

RESUMO

In vitro evaluations are essential to gaining a better understanding of re-osseointegration, while reducing animal use and the overall costs of peri-implantitis studies. This pilot study evaluated preosteoblast migration from 3-D-printed scaffolds to decontaminated titanium microimplants, creating a system that tries to mimic the bone-implant interface. Smooth (S) and minimally rough (R) titanium microimplants were incubated in Escherichia coli cultures and divided into six groups according to the decontamination protocol applied: EDTA gel (EDTA); chlorhexidine (CHL); chlorhexidine-soaked gauze (GCHL); scaling (SC); titanium brush (TiB); and implantoplasty (IP). Pristine S and R microimplants were used as the controls (C). After the decontamination procedures, the microimplants were inserted in 3-D-printed polyurethane-based scaffolds previously inoculated with preosteoblast cell cultures. Cellular migration was assessed after 24, 72 and 120 hours by ATP quantification. At the 120-hour time point, there was no statistically significant difference between S-C, S-EDTA, S-CHL, S-GCHL and S-SC (p > 0.05), and between R-C, R-EDTA and R-GCHL (p > 0.05). The in vitro model developed in this pilot study successfully demonstrated cell migration on the different decontaminated surfaces. This methodology suggests that on smooth microimplants, EDTA, GCHL, SC and TiB decontamination may have a reduced impact on preosteoblast migration, while on minimally rough microimplants, EDTA and GCHL decontamination affected cell migration the least. However, when selecting a decontamination protocol, the effectiveness of the decontamination per se must also be considered.


Assuntos
Peri-Implantite , Titânio , Animais , Clorexidina , Descontaminação , Projetos Piloto , Propriedades de Superfície , Titânio/farmacologia
4.
Int J Med Sci ; 18(13): 2971-2980, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34220325

RESUMO

NIR fluorescence imaging using bisphosphonate-Indocyanine green has been indicated for early interproximal caries detection. This study assessed diagnostic accuracy of caries detection by NIR fluorescence imaging with OsteoSense 750® (OS750) in vitro and ex vivo, and to analyze the therapeutic efficacy of a bisphosphonate (Etidronate) in inhibiting enamel caries progression in vitro. Methods: Four experiments were conducted using extracted human teeth; 1) to calculate the infiltration rate of OS750 into interproximal white spot lesions using fluorescence microscope, 2) to assess diagnostic accuracy of interproximal natural white spot lesions using desktop NIR fluorescence imaging device in vitro setting, 3) to assess diagnostic accuracy of artificially created deeper enamel carious lesion (0.5 mm~1.0 mm) using NIR fluorescence image through the head-mount display in ex vivo setting, 4) to compare the progression on the enamel caries lesions treated by Etidronate, NaF and distilled-water. Diagnostic accuracy was analyzed using sensitivity, specificity and receiver operating curves (ROC). The caries progression was calculated with micro-CT and was statistically analyzed using a two-way ANOVA and the Tukey HDS post-hoc test. Results: 1) The infiltration rate of OS750 was 101.83% ± 8.66 (Min: 90.10%, Max: 133.94%). 2) The average of sensitivity and specificity in vitro setting experiments were 86.7% ± 4.4% and 70% ± 11%, respectively. The average of area under the ROC curves (AUC) was 0.883 ± 0.059 indicating excellent performance. 3) The mean sensitivity and specificity in ex vivo setting was 82.97% ± 15% and 76.78% ± 13.27% respectively. 4) The carious lesion volume treated by Etidronate was significantly smaller at post treatment-1 (p<0.05) and treatment-2 (p<0.01) than the control. There was no significant difference in lesion volume in the Etidronate and NaF group at the time point of post treatment-1. Conclusion: This study suggests that bisphosphonates contribute to both early diagnosis of enamel caries and inhibition of caries progression.


Assuntos
Conservadores da Densidade Óssea/administração & dosagem , Meios de Contraste/administração & dosagem , Cárie Dentária/diagnóstico , Difosfonatos/administração & dosagem , Imagem Óptica/métodos , Cárie Dentária/tratamento farmacológico , Cárie Dentária/patologia , Esmalte Dentário/diagnóstico por imagem , Esmalte Dentário/efeitos dos fármacos , Esmalte Dentário/patologia , Progressão da Doença , Ácido Etidrônico/administração & dosagem , Fluorescência , Humanos , Sensibilidade e Especificidade , Fluoreto de Sódio/administração & dosagem , Microtomografia por Raio-X
5.
J Periodontol ; 92(5): 704-715, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-32946119

RESUMO

BACKGROUND: The objective of this study is to evaluate titanium decontamination after different protocols while assessing changes in surface roughness, chemical composition, and wettability. METHODS: Ninety-six smooth (S) and 96 minimally rough (R) titanium microimplants were used. Pristine microimplants were reserved for negative control (S-nC/R-nC, n = 9), while the remaining microimplants were incubated in Escherichia coli culture. Non-decontaminated microimplants were used as positive control (S-pC/R-pC, n = 3). The other microimplants were divided into seven different decontamination protocols (12 S/R per group): 24% EDTA, 2% chlorhexidine (CHL), gauze soaked in 2% chlorhexidine (GCHL), gauze soaked in ultrapure water (GMQ), scaling (SC), titanium brush (TiB), and implantoplasty (IP). Contaminated areas were assessed by scanning electron microscope images, chemical composition by energy dispersive X-ray spectroscopy, wettability by meniscus technique, and roughness by an optical profiler. RESULTS: Higher residual bacteria were observed in R-pC compared with S-pC (P <0.0001). When comparing S and R with their respective pC groups, the best results were obtained with GCHL, SC, TiB, and IP, with no difference between these protocols (P >0.05). Changes in surface roughness were observed after all treatments, with S/R-IP presenting the smoother and a less hydrophilic surface (P <0.05). Apart from IP protocol, all the other groups presented a more hydrophilic surface in R than in S microimplants (P <0.003). All decontamination protocols resulted in a lower percentage of superficial Ti when compared with S/R-nC (P <0.002). CONCLUSIONS: All decontamination protocols resulted in changes in roughness, wettability, and chemical composition, but GCHL, SC, TiB, an IP presented the best decontamination outcomes.


Assuntos
Descontaminação , Titânio , Bactérias , Clorexidina , Microscopia Eletrônica de Varredura , Propriedades de Superfície
6.
Int J Oral Sci ; 12(1): 36, 2020 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-33380730

RESUMO

The aim of this study was to evaluate the biological efficacy of a unique perpendicular protrusion of type-I collagen (Col-I) from TiO2 nanotubes (NT-EPF surface). We hypothesized that the NT-EPF surface would play bifunctional roles in stimulating platelet-mediated fibroblast recruitment and anchoring fibroblast-derived Col-I to form a perpendicular collagen assembly, mimicking the connective tissue attachment around natural teeth for the long-term maintenance of dental implants. Ti surface modification was accomplished in two steps. First, TiO2 nanotubes (NT) array was fabricated via anodization. Diameters and depths of NTs were controlled by applied voltage and duration. Subsequently, an electrophoretic fusion (EPF) method was applied to fuse Col-I into nanotube arrays in a perpendicular fashion. Surface wettability was assessed by contact angle measurement. The bioactivity of modified TiO2 surfaces was evaluated in terms of NIH3T3 fibroblast attachment, platelet activation, and collagen extension. Early attachment, aggregation, and activation of platelets as well as release of platelet-related growth factors were demonstrated on NT-EPF surfaces. Platelet-mediated NIH3T3 cells migration toward NT-EPF was significantly increased and the attached cells showed a typical fibrous morphology with elongated spindle shape. A direct linkage between pseudopod-like processes of fibroblasts to NT-EPF surfaces was observed. Furthermore, the engineered EPF collagen protrusion linked with cell-derived collagen in a perpendicular fashion. Within the limitation of this in vitro study, the TiO2 nanotube with perpendicular Col-I surface (NT-EPF) promoted better cell attachment, induced a strong platelet activation which suggested the ability to create a more robust soft tissue seal.


Assuntos
Colágeno Tipo I , Nanotubos , Animais , Adesão Celular , Camundongos , Células NIH 3T3 , Propriedades de Superfície , Titânio
7.
Arch Oral Biol ; 118: 104861, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32835988

RESUMO

OBJECTIVE: To show the possible occurrence of exosomal transport of neprilysin from masseter muscle to hippocampus via trigeminal nerve in the living mouse. DESIGN: Mouse C2C12 myotube-derived exosomes were labeled with near-infrared (NIR) dye and injected into the masseter muscle to track their fluorescence from masseter muscle to hippocampus via trigeminal nerve. A plasmid vector encoding green fluorescent protein (GFP)-tagged neprilysin (GFP-neprilysin) was transfected into masseter muscle of C57BL/6 J mice. Expression of mRNA and encoded protein of the transgene was identified in masseter muscle, trigeminal nerve and hippocampus by RT-PCR and Western blot, respectively. RESULTS: Peak of exosomal NIR in masseter muscle at time 0 rapidly reduced at 3 h and 6 h along with the subsequent increases in trigeminal nerve and hippocampus. Expression of GFP-neprilysin mRNA was detected in masseter muscle, but not trigeminal nerve and hippocampus. On the other hand, the corresponding protein of GFP-neprilysin was identified in the three tissues on day 3 after transfection into masseter muscle as a single band on Western blots with anti-GFP and anti-neprilysin antibodies. CONCLUSION: The appearance of GFP-neprilysin protein in trigeminal nerve and hippocampus without a corresponding mRNA expression indicated the protein's origin from the masseter muscle. Concomitant migration of NIR-exosomes from masseter muscle to hippocampus via trigeminal nerve suggested the possible occurrence of exosomal transport of neprilysin.


Assuntos
Hipocampo/metabolismo , Músculo Masseter/metabolismo , Neprilisina/metabolismo , Nervo Trigêmeo/metabolismo , Animais , Exossomos , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde , Camundongos , Camundongos Endogâmicos C57BL , Neprilisina/genética , Transporte Proteico
8.
J Nanobiotechnology ; 17(1): 34, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823919

RESUMO

Natural teeth are supported by connective tissue collagen fibers that insert perpendicularly in the tooth cementum. Perpendicular insertion plays an important role in the maintenance of the junction between the oral epithelium and the periodontal connective tissue. Most titanium dental implant surfaces have no micro or macro structure to support perpendicularly oriented collagen attachment. Without this tight biologic seal to resist bacterial invasion and epithelial downgrowth, progressive bone loss in peri-implantitis is seen around dental implants. The purpose of this study was to establish the perpendicularly oriented collagen attachment to titanium oxide nanotube (TNT), and to assess its binding stability. TNT was prepared on the titanium-surface by anodization. Scanning electron microscopy (SEM) showed a regularly aligned TNT with an average 67 nm-diameter when anodized at 30 V for 3 h. Subsequently, collagen type I (CoI) was electrophoretically fused to anodic TNT in native polyacrylamide gel system where negatively charged CoI-C term was perpendicularly navigated to TNT. SEM and atomic force microscopy (AFM) were used to analyze CoI on the TiO2 and TNT surface. Several tens of nanometers of CoI protrusion were recorded by AFM. These protrusions may be long enough to be priming sites for cell-secreted CoI. CoI laid parallel to the titanium surface when fused by a chemical linker. Binding resistance of CoI against drastic ultrasonication was measured by Fourier-transform infrared spectroscopy attenuated total reflection (FTIR-ATR). The electrophoretically fused CoI in the titanium nanotube (TNT-CoIEPF) showed the significantly greatest binding resistance than the other groups (P < 0.01, a 1-way ANOVA and Tukey HSD post hoc test). Furthermore, TNT-CoIEPF surface rejected epithelial cell stretching and epithelial sheet formation. Chemically linked horizontal CoI on titanium oxide (TiO2) facilitated epithelial cell stretching and sheet formation.


Assuntos
Colágeno Tipo I/química , Tecido Conjuntivo/química , Implantes Dentários , Nanotubos/química , Titânio/química , Materiais Biocompatíveis/química , Adesão Celular/efeitos dos fármacos , Técnicas Eletroquímicas , Células Epiteliais/efeitos dos fármacos , Humanos , Tamanho da Partícula , Propriedades de Superfície
9.
Neurosci Lett ; 698: 180-185, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30639512

RESUMO

Although the effects of neprilysin (NEP), also called CD10, on the clearance of Alzheimer's disease (AD)-associated amyloid-ß (Aß) have been reported, NEP is not made in the brain, and the mechanism for the transport of NEP to the brain has not been investigated. Our hypothesis is that muscle packages NEP in exosomes in response to a neuromuscular signal and sends it to the brain via retrograde axonal transport. The masseter muscle (MM) and the trigeminal nerve (TGN) are good candidates for this mechanism by virtue of their proximity to the brain. The aim of this study was to trace the NEP protein from the MM, through the TGN, and to the hippocampus (HPC) in muscle contraction models in vitro and in vivo. NEP expression in mouse tissue lysates was analyzed by RT-PCR and Western blot. Four-week-old mice were perfused to remove blood NEP contamination. The MM expressed substantial levels of NEP protein and mRNA. On the other hand, a remarkably high level of NEP protein was measured in the TGN in the absence of mRNA. NEP protein, without the corresponding mRNA, was also detected in the HPC. These results suggested that the MM derived NEP was taken up by the TGN, which in turn permitted NEP access to the central nervous system and within it the HPC. When the MM was induced to contract by electric stimulation in freshly euthanized mice, NEP protein decreased in the MM in a stimulus time-dependent manner, while that in the TGN and the HPC increased sequentially. Furthermore, NIR-labeled exosomes tracked along the same route. Finally, carbachol induced secretion of exosomal NEP in C2C12-derived myotube cells. These results support our hypothesis that MM-derived NEP is transported along the TGN to reach the HPC following electrical or cholinergic stimulation.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Hipocampo/metabolismo , Músculo Masseter/metabolismo , Neprilisina/metabolismo , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Neurônios/metabolismo
10.
Int J Oral Sci ; 10(3): 21, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29961761

RESUMO

Epithelial attachment via the basal lamina on the tooth surface provides an important structural defence mechanism against bacterial invasion in combating periodontal disease. However, when considering dental implants, strong epithelial attachment does not exist throughout the titanium-soft tissue interface, making soft tissues more susceptible to peri-implant disease. This study introduced a novel synthetic peptide (A10) to enhance epithelial attachment. A10 was identified from a bacterial peptide display library and synthesized. A10 and protease-activated receptor 4-activating peptide (PAR4-AP, positive control) were immobilized on commercially pure titanium. The peptide-treated titanium showed high epithelial cell migration ability during incubation in platelet-rich plasma. We confirmed the development of dense and expanded BL (stained by Ln5) with pericellular junctions (stained by ZO1) on the peptide-treated titanium surface. In an adhesion assay of epithelial cells on A10-treated titanium, PAR4-AP-treated titanium, bovine root and non-treated titanium, A10-treated titanium and PAR4-AP-treated titanium showed significantly stronger adhesion than non-treated titanium. PAR4-AP-treated titanium showed significantly higher inflammatory cytokine release than non-treated titanium. There was no significant difference in inflammatory cytokine release between A10-treated and non-treated titanium. These results indicated that A10 could induce the adhesion and migration of epithelial cells with low inflammatory cytokine release. This novel peptide has a potentially useful application that could improve clinical outcomes with titanium implants and abutments by reducing or preventing peri-implant disease.


Assuntos
Benzenoacetamidas/farmacologia , Adesão Celular/efeitos dos fármacos , Inserção Epitelial/efeitos dos fármacos , Células Epiteliais/citologia , Piperidonas/farmacologia , Titânio/química , Sequência de Aminoácidos , Animais , Benzenoacetamidas/síntese química , Bovinos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Implantes Dentários , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Varredura , Piperidonas/síntese química , Plasma Rico em Plaquetas , Receptores de Trombina , Propriedades de Superfície
11.
J Clin Dent ; 28(3): 62-64, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29211953

RESUMO

OBJECTIVES: This study aims to assess the difference in microbial contamination of a toothbrush with a smooth handle versus a toothbrush with a grooved handle design. METHODS: Twenty-six volunteers were randomized into two groups. The first group used a smooth handle toothbrush for two months, followed by a grooved handle toothbrush for two months. The second group had the order reversed. Following the two-month use, the toothbrushes were submitted for microbial analysis. Effect size, as well as Wilcoxon signed-rank test were used to calculate the differences between total colony count, bacterial DNA, and endotoxin levels from the two toothbrush handle types. RESULTS: There was no significant difference in colony count between the smooth (mean 580 CFU/mL, SD 1,684 CFU/mL) and grooved (mean 19,059 CFU/mL, SD 80,972 CFU/mL) handles (p = 0.12). Total DNA count was significantly less (p = 0.01) on the smooth handle (mean 68,038 RFU/mL, SD 81,659) compared to the grooved handle (mean 209,312 RFU/mL, SD 257,169 RFU/mL). Endotoxin levels were significantly less (p = 0.01) on the smooth handle (mean 0.16 EU/mL, SD 0.30 EU/mL) compared to the grooved handle (mean 0.43 EU/mL, SD 0.49 EU/mL). CONCLUSIONS: The smooth handle toothbrush had significantly less bacterial contamination compared to the grooved handle toothbrush, as measured by total DNA count and endotoxin levels.


Assuntos
Placa Dentária , Escovação Dentária , Contagem de Colônia Microbiana , Placa Dentária/microbiologia , Placa Dentária/prevenção & controle , Índice de Placa Dentária , Desenho de Equipamento , Humanos , Escovação Dentária/instrumentação
12.
Bioorg Med Chem Lett ; 27(3): 393-397, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28057422

RESUMO

Aberrant activation of mast cells contributes to the development of numerous diseases including cancer, autoimmune disorders, as well as diabetes and its complications. The influx of extracellular calcium via the highly calcium selective calcium-release activated calcium (CRAC) channel controls mast cell functions. Intracellular calcium homeostasis in mast cells can be maintained via the modulation of the CRAC channel, representing a critical point for therapeutic interventions. We describe the structure-activity relationship study (SAR) of indazole-3-carboxamides as potent CRAC channel blockers and their ability to stabilize mast cells. Our SAR results show that the unique regiochemistry of the amide linker is critical for the inhibition of calcium influx, the release of the pro-inflammatory mediators ß-hexosaminidase and tumor necrosis factor α by activated mast cells. Thus, the indazole-3-carboxamide 12d actively inhibits calcium influx and stabilizes mast cells with sub-µM IC50. In contrast, its reverse amide isomer 9c is inactive in the calcium influx assay even at 100µM concentration. This requirement of the specific 3-carboxamide regiochemistry in indazoles is unprecedented in known CRAC channel blockers. The new structural scaffolds described in this report expand the structural diversity of the CRAC channel blockers and may lead to the discovery of novel immune modulators for the treatment of human diseases.


Assuntos
Amidas/química , Bloqueadores dos Canais de Cálcio/química , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Amidas/síntese química , Amidas/farmacologia , Bloqueadores dos Canais de Cálcio/síntese química , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/química , Humanos , Indazóis/química , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa
13.
PLoS One ; 11(10): e0164693, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27741287

RESUMO

The aim of this study was to produce epithelial attachment on a typical implant abutment surface of smooth titanium. A challenging complication that hinders the success of dental implants is peri-implantitis. A common cause of peri-implantitis may results from the lack of epithelial sealing at the peri-implant collar. Histologically, epithelial sealing is recognized as the attachment of the basement membrane (BM). BM-attachment is promoted by activated platelet aggregates at surgical wound sites. On the other hand, platelets did not aggregate on smooth titanium, the surface typical of the implant abutment. We then hypothesized that epithelial BM-attachment was produced when titanium surface was modified to allow platelet aggregation. Titanium surfaces were coated with a protease activated receptor 4-activating peptide (PAR4-AP). PAR4-AP coating yielded rapid aggregation of platelets on the titanium surface. Platelet aggregates released robust amount of epithelial chemoattractants (IGF-I, TGF-ß) and growth factors (EGF, VEGF) on the titanium surface. Human gingival epithelial cells, when they were co-cultured on the platelet aggregates, successfully attached to the PAR4-AP coated titanium surface with spread laminin5 positive BM and consecutive staining of the epithelial tight junction component ZO1, indicating the formation of complete epithelial sheet. These in-vitro results indicate the establishment of epithelial BM-attachment to the titanium surface.


Assuntos
Peptídeos/química , Receptores de Trombina/metabolismo , Titânio/química , Plaquetas/citologia , Plaquetas/metabolismo , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Materiais Revestidos Biocompatíveis/farmacologia , Citocinas/metabolismo , Implantes Dentários , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Gengiva/citologia , Humanos , Imuno-Histoquímica , Microscopia Eletrônica de Varredura , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Receptores de Trombina/química , Propriedades de Superfície , Proteína da Zônula de Oclusão-1/metabolismo , Calinina
14.
Biochem Biophys Res Commun ; 459(1): 71-4, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25721672

RESUMO

Bone matrix provides unknown essential cues for osteoblast lineage cells to develop, grow, repair and remodel bones via adherent plasma membrane. Because of its tight sealing with bone matrix in vivo and culture surface in vitro as well, the adherent plasma membrane has been unveiled target of investigation to date. Herein, we report a new approach to explore the adherence plasma membrane of osteoblasts with biofunctional peptide candidates in a bacterial peptide library. To accomplish this, human osteoblast like hFOB 1.19 cells were cultured on porous filter with 8 µm pore through which bacterial peptides were allowed to meet the membrane for affinity selection. The affinity-selected peptides were coated on culture plate to further evaluate their influence on osteoblastic cell adhesion, as well as expressions of osteoblast differentiation markers, alkaline phosphatase and osteocalcin. Finally, the serial screenings identified two prominent active peptides that enhanced the differentiation markers nearly to the same level as a control peptide of bone morphogenetic protein-2. Osteogenic activity is expected for the peptides when immobilized on bone implant surface.


Assuntos
Membrana Celular/efeitos dos fármacos , Peptídeos/farmacologia , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Anabolizantes/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Dados de Sequência Molecular , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Biblioteca de Peptídeos
15.
Mol Cancer Res ; 12(7): 1042-54, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24784839

RESUMO

UNLABELLED: Activating mutations and/or overexpression of FGFR3 are common in bladder cancer, making FGFR3 an attractive therapeutic target in this disease. In addition, FGFR3 gene rearrangements have recently been described that define a unique subset of bladder tumors. Here, a selective HSP90 inhibitor, ganetespib, induced loss of FGFR3-TACC3 fusion protein expression and depletion of multiple oncogenic signaling proteins in RT112 bladder cells, resulting in potent cytotoxicity comparable with the pan-FGFR tyrosine kinase inhibitor BGJ398. However, in contrast to BGJ398, ganetespib exerted pleiotropic effects on additional mitogenic and survival pathways and could overcome the FGFR inhibitor-resistant phenotype of FGFR3 mutant-expressing 97-7 and MHG-U3 cells. Combinatorial benefit was observed when ganetespib was used with BGJ398 both in vitro and in vivo. Interestingly, two additional FGFR3 fusion-positive lines (RT4 and SW480) retained sensitivity to HSP90 inhibitor treatment by the ansamycins 17-AAG and 17-DMAG yet displayed intrinsic resistance to ganetespib or AUY922, both second-generation resorcinol-based compounds. Both cell lines, compared with RT112, expressed considerably higher levels of endogenous UGT1A enzyme; this phenotype resulted in a rapid glucuronidation-dependent metabolism and subsequent efflux of ganetespib from SW780 cells, thus providing a mechanism to account for the lack of bioactivity. IMPLICATIONS: Pharmacologic blockade of the molecular chaperone HSP90 represents a promising approach for treating bladder tumors driven by oncogenic gene rearrangements of FGFR3. Furthermore, UDP-glucuronosyltransferase enzyme expression may serve as a predictive factor for clinical response to resorcinol-based HSP90 inhibitors.


Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Triazóis/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Distribuição Aleatória , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Triazóis/farmacocinética , Neoplasias da Bexiga Urinária/genética , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Clin Cancer Res ; 20(2): 413-24, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24173541

RESUMO

PURPOSE: Treatment options for patients with triple-negative breast cancer (TNBC) are largely limited to systemic chemotherapies, which have shown disappointing efficacy in the metastatic setting. Here, we undertook a comprehensive evaluation of the activity of ganetespib, a potent inhibitor of HSP90, in this malignancy. EXPERIMENTAL DESIGN: The antitumor and antimetastatic activity of ganetespib was investigated using TNBC cell lines and xenograft models. Combinatorial drug analyses were performed with chemotherapeutic agents and concomitant effects on DNA damage and cell-cycle disruption were assessed in vitro; antitumor efficacy was assessed in vivo. Metabolic and objective tumor responses were evaluated in patients with metastatic TNBC undergoing ganetespib treatment. RESULTS: Ganetespib simultaneously deactivated multiple oncogenic pathways to potently reduce cell viability in TNBC cell lines, and suppressed lung metastases in experimental models. Ganetespib potentiated the cytotoxic activity of doxorubicin via enhanced DNA damage and mitotic arrest, conferring superior efficacy to the doxorubicin-cyclophosphamide regimen in TNBC xenografts. Ganetespib also promoted mitotic catastrophe and apoptosis in combination with taxanes in vitro, and these effects translated to significantly improved combinatorial activity in vivo. Marked tumor shrinkage of metastatic lung and lymphatic lesions were seen in patients on ganetespib monotherapy. CONCLUSION: The preclinical activity profile and clinical evidence of tumor regression suggest that ganetespib offers considerable promise as a new therapeutic candidate to target TNBC.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Triazóis/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Antineoplásicos/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Mitose/efeitos dos fármacos , Metástase Neoplásica , Estadiamento de Neoplasias , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios X , Triazóis/uso terapêutico , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Free Radic Biol Med ; 52(10): 2142-50, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22542443

RESUMO

Elesclomol is an investigational drug that exerts potent anticancer activity through the elevation of reactive oxygen species (ROS) levels and is currently under clinical evaluation as a novel anticancer therapeutic. Here we report the first description of selective mitochondrial ROS induction by elesclomol in cancer cells based on the unique physicochemical properties of the compound. Elesclomol preferentially chelates copper (Cu) outside of cells and enters as elesclomol-Cu(II). The elesclomol-Cu(II) complex then rapidly and selectively transports the copper to mitochondria. In this organelle Cu(II) is reduced to Cu(I), followed by subsequent ROS generation. Upon dissociation from the complex, elesclomol is effluxed from cells and repeats shuttling elesclomol-Cu complexes from the extracellular to the intracellular compartments, leading to continued copper accumulation within mitochondria. An optimal range of redox potentials exhibited by copper chelates of elesclomol and its analogs correlated with the elevation of mitochondrial Cu(I) levels and cytotoxic activity, suggesting that redox reduction of the copper triggers mitochondrial ROS induction. Importantly the mitochondrial selectivity exhibited by elesclomol is a distinct characteristic of the compound that is not shared by other chelators, including disulfiram. Together these findings highlight a unique mechanism of action with important implications for cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Cobre/metabolismo , Hidrazinas/farmacologia , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Cobre/química , Humanos , Mitocôndrias/efeitos dos fármacos , Neoplasias/metabolismo , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
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